7TM receptor (H2CAA71)

ABSTRACT

Novel 7TM receptor (H2CAA71) polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Novel 7TM receptor (H2CAA71) polypeptides and polynucleotides in the design of protocols for the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson&#39;s disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington&#39;s disease or Gilles dela Tourett&#39;s syndrome, among others and diagnostic assays for such conditions.

This application is a division of U.S. application Ser. No. 08/866,757,filed May 30, 1997 now U.S. Pat. No. 5,858,716.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to G-protein coupledreceptor family, hereinafter referred to as Novel 7TM receptor(H2CAA71). The invention also relates to inhibiting or activating theaction of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al.,Science, 1987, 238:650-656; Bunzow, J. R., et al., Nature, 1988,336:783-787), G-proteins themselves, effector proteins, e.g.,phospholipase C, adenyl cyclase, and phosphodiesterase, and actuatorproteins, e.g., protein kinase A and protein kinase C (Simon, M. I., etal., Science, 1991, 252:802-8).

For example, in one form of signal transduction, the effect of hormonebinding is activation of the enzyme, adenylate cyclase, inside the cell.Enzyme activation by hormones is dependent on the presence of thenucleotide GTP. GTP also influences hormone binding. A G-proteinconnects the hormone receptor to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by a hormone receptor. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself, returns the G-proteinto its basal, inactive form. Thus, the G-protein serves a dual role, asan intermediate that relays the signal from receptor to effector, and asa clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors hasbeen characterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane α-helices connected byextracellular or cytoplasmic loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

G-protein coupled receptors (otherwise known as 7TM receptors) have beencharacterized as including these seven conserved hydrophobic stretchesof about 20 to 30 amino acids, connecting at least eight divergenthydrophilic loops. The G-protein family of coupled receptors includesdopamine receptors which bind to neuroleptic drugs used for treatingpsychotic and neurological disorders. Other examples of members of thisfamily include, but are not limited to, calcitonin, adrenergic,endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,histamine, thrombin, kinin, follicle stimulating hormone, opsins,endothelial differentiation gene-1, rhodopsins, odorant, andcytomegalovirus receptors.

Most G-protein coupled receptors have single conserved cysteine residuesin each of the first two extracellular loops which form disulfide bondsthat are believed to stabilize functional protein structure. The 7transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6,and TM7. TM3 has been implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) ofcysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theβ-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said socket beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form a polar ligand binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331)Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7transmembrane (7 TM) receptors have been successfully introduced ontothe market.

This indicates that these receptors have an established, proven historyas therapeutic targets. Clearly there is a need for identification andcharacterization of further receptors which can play a role inpreventing, ameliorating or correcting dysfunctions or diseases,including, but not limited to, infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; pain; cancers, anorexia; bulimia; asthma; Parkinson's disease;acute heart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to Novel 7TM receptor (H2CAA71)polypeptides and recombinant materials and methods for their production.Another aspect of the invention relates to methods for using such Novel7TM receptor (H2CAA71) polypeptides and polynucleotides. Such usesinclude the treatment of infections such as bacterial, fungal, protozoanand viral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others. Instill another aspect, the invention relates to methods to identifyagonists and antagonists using the materials provided by the invention,and treating conditions associated with Novel 7TM receptor (H2CAA71)imbalance with the identified compounds. Yet another aspect of theinvention relates to diagnostic assays for detecting diseases associatedwith inappropriate Novel 7TM receptor (H2CAA71) activity or levels.

DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Novel 7TM receptor (H2CAA71)” refers, among others, to a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2, or anallelic variant thereof.

“Receptor Activity” or “Biological Activity of the Receptor” refers tothe metabolic or physiologic function of said Novel 7TM receptor(H2CAA71) including similar activities or improved activities or theseactivities with decreased undesirable side-effects. Also included areantigenic and immunogenic activities of said Novel 7TM receptor(H2CAA71).

“Novel 7TM receptor (H2CAA71) gene” refers to a polynucleotidecomprising the nucleotide sequence set forth in SEQ ID NO:1 or allelicvariants thereof and/or their complements.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

“Identify” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994p; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO: 1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3 terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% identity to a reference amino acid sequence ofSEQ ID NO:2 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO: 2. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to Novel 7TM receptor(H2CAA71) polypeptides. The Novel 7TM receptor (H2CAA71) polypeptidesinclude the polypeptide of SEQ ID NO:2; as well as polypeptidescomprising the amino acid sequence of SEQ ID NO:2; and polypeptidescomprising the amino acid sequence which have at least 80% identity tothat of SEQ ID NO:2 over its entire length, and still more preferably atleast 90% identity, and even still more preferably at least 95% identityto SEQ ID NO: 2. Furthermore, those with at least 97-99% are highlypreferred. Also included within Novel 7TM receptor (H2CAA71)polypeptides are polypeptides having the amino acid sequence which haveat least 80% identity to the polypeptide having the amino acid sequenceof SEQ ID NO: 2 over its entire length, and still more preferably atleast 90% identity, and even still more preferably at least 95% identityto SEQ ID NO: 2. Furthermore, those with at least 97-99% are highlypreferred. Preferably Novel 7TM receptor (H2CAA71) polypeptides exhibitat least one biological activity of the receptor.

The Novel 7TM receptor (H2CAA71) polypeptides may be in the form of the“mature” protein or may be a part of a larger protein such as a fusionprotein. It is often advantageous to include an additional amino acidsequence which contains secretory or leader sequences, pro-sequences,sequences which aid in purification such as multiple histidine residues,or an additional sequence for stability during recombinant production.

Fragments of the Novel 7TM receptor (H2CAA71) polypeptides are alsoincluded in the invention. A fragment is a polypeptide having an aminoacid sequence that entirely is the same as part, but not all, of theamino acid sequence of the aforementioned Novel 7TM receptor (H2CAA71)polypeptides. As with Novel 7TM receptor (H2CAA71) polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof Novel 7TM receptor (H2CAA71) polypeptide. In this context “about”includes the particularly recited ranges larger or smaller by several,5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of Novel 7TM receptor (H2CAA71) polypeptides,except for deletion of a continuous series of residues that include theamino terminus, or a continuous series of residues that includes thecarboxyl terminus or deletion of two continuous series of residues, oneincluding the amino terminus and one including the carboxyl terminus.Also preferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions. Otherpreferred fragments are biologically active fragments. Biologicallyactive fragments are those that mediate receptor activity, includingthose with a similar activity or an improved activity, or with adecreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biologicalactivity of the receptor, including antigenic activity. Variants of thedefined sequence and fragments also form part of the present invention.Preferred variants are those that vary from the referents byconservative amino acid substitutions—i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The Novel 7TM receptor (H2CAA71) polypeptides of the invention can beprepared in any suitable manner. Such polypeptides include isolatednaturally occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

Polynucleotides of the Invention

Another aspect of the invention relates to Novel 7TM receptor (H2CAA71)polynucleotides. Novel 7TM receptor (H2CAA71) polynucleotides includeisolated polynucleotides which encode the Novel 7TM receptor (H2CAA71)polypeptides and fragments, and polynucleotides closely related thereto.More specifically, Novel 7TM receptor (H2CAA71) polynucleotide of theinvention include a polynucleotide comprising the nucleotide sequenceset forth in SEQ ID NO:1 encoding a Novel 7TM receptor (H2CAA71)polypeptide of SEQ ID NO: 2, and polynucleotide having the particularsequence of SEQ ID NO:1. Novel 7TM receptor (H2CAA71) polynucleotidesfurther include a polynucleotide comprising a nucleotide sequence thathas at least 80% identity to a nucleotide sequence encoding the Novel7TM receptor (H2CAA71) polypeptide of SEQ ID NO:2 over its entirelength, and a polynucleotide that is at least 80% identical to thathaving SEQ ID NO:1 over its entire length. In this regard,polynucleotides at least 90% identical are particularly preferred, andthose with at least 95% are especially preferred. Furthermore, thosewith at least 97% are highly preferred and those with at least 98-99%are most highly preferred, with at least 99% being the most preferred.Also included under Novel 7TM receptor (H2CAA71) polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO:1 to hybridize under conditions useablefor amplification or for use as a probe or marker. The invention alsoprovides polynucleotides which are complementary to such Novel 7TMreceptor (H2CAA71) polynucleotides.

Novel 7TM receptor (H2CAA71) of the invention is structurally related toother proteins of the G-protein coupled receptor family, as shown by theresults of sequencing the cDNA of Table 1 (SEQ ID NO:1) encoding humanNovel 7TM receptor (H2CAA71). The cDNA sequence of SEQ ID NO:1 containsan open reading frame (nucleotide numbers 483 to 2415) encoding apolypeptide of 644 amino acids (SEQ ID NO:2). The amino acid sequence ofTable 2 (SEQ ID NO:2) has about 27% identity (using FASTA) in 571 aminoacid residues with Bovine follicle stimulating hormone receptor. Houde A1994 Mol. Reprod. Dev., 39, 127-135. The nucleotide sequence of Table 1(SEQ ID NO:1) has about 60% identity (using FASTA) in 80 nucleotideresidues with Bovine follicle stimulating hormone receptor. Houde A 1994Mol. Reprod. Dev., 39, 127-135.

TABLE 1^(a)    1 GCTTCCATCC TAATACAACT CACTATAGGG CTCGAGCGGC CGCCCGGGCA  51 GGTGCTTGAC GGAGGTGCCT GTGCACCCCC TCAGCAATCT GCCCACCCTA  101CAGGCGCTGA CCCTGGCTCT CAACAAgATC TCAAGCATCC CTgACTTTGC  151 ATTTACCAACCTTTCAAGCC TGGTAgTTCT GCATCTTCAT AACAATAAAA  201 TTAgAAGCCT GAGTCAACACTGTTTTGATG GACTAgATaA CCTGGAGACC  251 TTAgACTTGA ATTATAATAA CTTGGGGGAATTTCCTCAGG CTATTAAAGC  301 CCTTCCTAGC CTTAAAgAGC TAGGATTTCA TAGTAATTCTATTTCTGTTA  351 TCCCTATGGA GCATTTGATG GTAATCCACT CTTAAgAACT ATACATTTGT 401 ATGATAATCC TCTGTCTTTT GTGGGGAACT CAGCATTTCA CAAttTATCT  451GATCTTCATT CCCTAGTCAT TcGTGGTGCA AGCATGGTGC AGCAGTTCCC  501 CAATCTTACAGGAACTGTCC ACCTGGAAAG TCTGACTTTG ACAGGTACAA  551 AGATAAGCAG CATACCTAATAATTTGTGTC AAGAACAAAA GATGCTTAGG  601 ACTTTGGACT TGTCTTACAA TAATATAAGAGACCTTCCAA GTTTTAATGG  651 TTGCCATGCT CTGGAAGAAA TTTCTTTACA GCGTAATCAAATCTACCAAA  701 TAAAGGAAGG CACCTTTCAA GGCCTGATAT CTCTAAGGAT TCTAGATCTG 751 AGTAGAAACC TGATACATGA AATTCACAGT AGAGCTTTTG CCACACTTGG  801GCCAATAAct AACcTAGAtG TAAGTTTCAA tGAATTAACT TCcTTTCCtA  851 CGGAAGGCCtGAATGGGcTA AATCAACTGA AACTGGTGGG CAACTTCAAG  901 cTGAAAGAAG CCTTAGCAGCAAAAGACTTt GTTAACCTCa GGTCTTTATC  951 AGTACCATAT GCTTATCAGT GCTGTGCATTTtGGGGTTGT GAcTCTTATG 1001 CAAATTTAAA CACAGAAGAT AACAGCCTCC AGGACCACAGTGTGGCACAG 1051 GAGAAAGGTA CTGCTGATGC AGCAAATGTC ACAAGCACTC TTGAAAATGA1101 AGAACATAGT CAAATAATTA TCCATTGTAC ACCTTCAACA GGTGCTTTTA 1151AGCCCTGTGA ATATTTACTG GGAAGCTGGA TGATTCGTCT TACTGTGTGG 1201 TTCATTTTCTTGGTTGCATT ATTTTTCAAC CTGCTTGTTA TTTTAACAAC 1251 ATTTGCATCT TGTACATCACTGCCTTCGTC CAAATTGTTT ATAGGCTTGA 1301 TTTCTGTGTC TAACTTATTC ATGGGAATCTATACTGGCAT CCTAACTTTT 1351 CTTGATGCTG TGTCCTGGGG CAGATTCGCT GAATTTGGCATTTGGTGGGA 1401 AACTGGCAGT GGCTGCAAAG TAACTGGGTT TCTTGCAgTT TTCTCCTCAG1451 AAAGTGCCAT ATTTTTATTA ATGCTAgCAA CTGTCGAAAG AAgCTTATCT 1501GCAAAAGATA TAATGAAAAA TGGGAAGAGC AATCATCTCA AACAGTTCCG 1551 GGTTGCTGCCCTTTTGGCTT TCCTAGGTGC TACAGTAACA GGCTGTTTTC 1601 CCCTTTTCCA TAGAGGGGAATATTCTGCAT CACCCCTTTG TTTGCCATTT 1651 CCTACAGGTG AAACGCCATC ATTAGGATTCACTGTAACGT TAGTGCTATT 1701 AAACTCACTA GCATTTTTAT TAATGGCCGT TATCTACACTAAGCTATACT 1751 GCAACTTGGA AAAAGAGGAC CTCTCAGAAA ACTCACAATC TAGCATGATT1801 AAGCATGTCG CTTGGCTAAT CTTCACCAAT TGCATCTTTT TCTGCCCTGT 1851GGCGTTTTTT TCATTTGCAC CATTGATCAC TGCAATCTCT ATCAGCCCCG 1901 AAATAATGAAGTCTGTTACT CTGATATTTT TTCCATTGCC TGCTTGCCTG 1951 AATCCAGTCC TGTATGTTTTCTTCAACCCA AAGTTTAAAG AAGACTGGAA 2001 GTTACTGAAG CGACGTGTTA CCAAGAAAAGTGGATCAGTT TCAGTTTCCA 2051 TCAGTAGCCA AGGTGGTTGT CTGGAACAGG ATTTCTACTACGACTGTGGC 2101 ATGTACTCAC ATTTGCAGGG CAACCTGACT GTTTGCGACT GCTGCGAATC2151 GTTTCTTTTA ACAAAGCCAG TATCATGCAA ACACTTGATA AAATCACACA 2201GCTGTCCTGC ATTGGCAGTG GCTTCTTGCC AAAGACCTGA GGGCTACTGG 2251 TCCGACTGTGGCACACAGTC GGCCCACTCT GATTATGCAG ATGAAGAAGA 2301 TTCCTTTGTC TCAGACAGTTCTGACCAGGT GCAGGCCTGT GGACGAGCCT 2351 GCTTCTACCA GAGTAGAGGA TTCCCTTTGGTGCGCTATGC TTACAATCTA 2401 CCAAGAGTTA AAGACTGAAC TACTGTGTGT GTAACCGTTTCCCCCGTCAA 2451 CCAAAATCAG TGTTTATAGA GTGAACCCTA TTCTCATCTT TCATCTGGGA2501 AGCACTTCTG TAATCACTGC CTGGTGTCAC TTAGAAGAAG GAGAGGTGGC 2551AGTTTATTTC TCAAACCAGT CATTTTCAAA GAACAGGTGC CTAAATTATA 2601 AATTGGTGAAAAATGCAATG TCCAAGCAAT GTATGATCTG TTTGAAACAA 2651 ATATATGACT TGAAAAGGATCTTAGGTGTA GTAGAGCAAT ATAATGTTAG 2701 TTTTTTCTGA TCCATAAGAA GCAAATTTATACCTATTTGT GTATTAAGCA 2751 CAAGATAAAG AACAGCTGTT AATATTTTTT AAAAATCTATTTTAAAATGT 2801 GATTTTCTAT AACTGAAGAA AATATCTTGC TAATTTTACC TAATGTTTCA2851 TCCTTAATCT CAGGACAACT TACTGCAGGG CCAAAAAAGG GACTGTCCCA 2901GCTAGAACTG TGAGAGTATA CATAGGCATT ACTTTATTAT GTTTTCACTT 2951 GCCATCCTTGACATAAGAGA ACTATAAATT TTGTTTAAGC AATTTATAAA 3001 TCTAAAACCT GAAGATGTTTTTAAAACAAT ATTAACAGCT GTTAGGTTAA 3051 AAAAATAGCT GGACATTTGT TTTCAGTCATTATACATTGC TTTGGTCCAA 3101 TCAGTAATTT TTTCTTAAGT GTTTTGTGAT TACACTACTAGAAAAAAAGT 3151 AAAAGGCTAA TTGCTGTGTG GGTTTAGTCG ATTTGGCTAA ACTACTAACT3201 AATGTGGGGG TTTAATAGTA TCTGAGGGAT TTGGTGGCTT CATGTAATGT 3251TCTCATTAAT GAATACTTCC TAATATCGTT GGCTCTACTA ATATTTTCCA 3301 ATTTGCTGGGATGTCACCTA GCAATAGCTT GGATTATATA GAAAGTAAAC 3351 TGTGGTCAAT ACTTGCATTTAATTAGACGA AACGGGGAGT AATTATGACA 3401 CGAAGTACTT ATGTTTATTT CTTAGTGAGCTGGATTATCT TGAACCTGTG 3451 CTATTAAATG GAAATTTCCA TACATCTTCC CCATACTATTTTTTATAAAA 3501 GAGCCTATTC AATAGCTCAG AGGTTGAACT CTGGTTAAAC AAGATAATAT3551 GTTATTAATA AAAATAGAAG AAGAAAGAAT AAAGCTTAGT CCTGTGTCTT 3601TAAAAATTAA AAATTTTACT TGATTCCCAT CTATGGGCTT TAGACCTATT 3651 ACTGGGTGGAGTCTTAAAGT TATAATTGTT CAATATGTTT TTTGAACAGT 3701 GTGCTAAATC AATAGCAAACCCACTGCCAT ATTAGTTATT CTGAATATAC 3751 TAAAAAAATC CAGCTAGATT GCAGTTTAATAATTAAACTG TACATACTGT 3801 GCATATAATG AATTTTTATC TTATGTAAAT TATTTTTAGAACACAAGTTG 3851 GGAAATGTGG CTTCTGTTCA TTTCGTTTAA TTAAAGCTAC CTCCTAAACT3901 ATAGTGGCTG CCAGTAGCAG ACTGTTAAAT TGTGGTTTAT ATACTTTTTG 3951CATTGTAAAT AGTCTTTGTT GTACATTGTC AGTGTAATAA AAACAGAATC 4001 TTTGTATATCAAAATCATGT AGTTTGTATA AAATGTGGGA AGGATTTATT 4051 TACAGTGTGT TGTAATTTTGTAAGGCCAAC TATTTACAAG TTTTAAAAAT 4101 TGCTATCATG TATATTTACA CATCTGATAAATATTAAATC ATAACTTGGT 4151 AAGAAACTCC TAATTAAAAG GTTTTTTCCA AAAAAAAAAAAAAAAAAAAA 4201 AAA ^(a)A nucleotide sequence of a human Novel 7TMreceptor (H2CAA71) (SEQ ID NO: 1).

TABLE 2^(b)   1 MVQQFPNLTG TVHLESLTLT GTKISSIPNN LCQEQKMLRT LDLSYNNIRD 51 LPSFNGCHAL EEISLQRNQI YQIKEGTFQG LISLRILDLS RNLIHEIHSR 101AFATLGPITN LDVSFNELTS FPTEGLNGLN QLKLVGNFKL KEALAAKDFV 151 NLRSLSVPYAYQCCAFWGCD SYANLNTEDN SLQDHSVAQE KGTADAANVT 201 STLENEEHSQ IIIHCTPSTGAFKPCEYLLG SWMIRLTVWF IFLVALFFNL 251 LVILTTFASC TSLPSSKLFI GLISVSNLFMGIYTGILTFL DAVSWGRFAE 301 FGIWWETGSG CKVTGFLAVF SSESAIFLLM LATVERSLSAKDIMKNGKSN 351 HLKQFRVAAL LAFLGATVTG CFPLFHRGEY SASPLCLPFP TGETPSLGFT401 VTLVLLNSLA FLLMAVIYTK LYCNLEKEDL SENSQSSMIK HVAWLIFTNC 451IFFCPVAFFS FAPLITAISI SPEIMKSVTL IFFPLPACLN PVLYVFFNPK 501 FKEDWKLLKRRVTKKSGSVS VSISSQGGCL EQDFYYDCGM YSHLQGNLTV 551 CDCCESFLLT KPVSCKHLIKSHSCPALAVA SCQRPEGYWS DCGTQSAHSD 601 YADEEDSFVS DSSDQVQACG RACFYQSRGFPLVRYAYNLP RVKD ^(b)An amino acid sequence of a human Novel 7TM receptor(H2CAA71) (SEQ ID NO: 2).

One polynucleotide of the present invention encoding Novel 7TM receptor(H2CAA71) may be obtained using standard cloning and screening, from acDNA library derived from mRNA in cells of Human placenta using theexpressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

The nucleotide sequence encoding Novel 7TM receptor (H2CAA71)polypeptide of SEQ ID NO:2 may be identical to the polypeptide encodingsequence contained in Table 1 (nucleotide number 483 to 2415 of SEQ IDNO:1), or it may be a sequence, which as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:2.

When the polynucleotides of the invention are used for the recombinantproduction of Novel 7TM receptor (H2CAA71) polypeptide, thepolynucleotide may include the coding sequence for the maturepolypeptide or a fragment thereof, by itself, the coding sequence forthe mature polypeptide or fragment in reading frame with other codingsequences, such as those encoding a leader or secretory sequence, apre-, or pro- or prepro-protein sequence, or other fusion peptideportions. For example, a marker sequence which facilitates purificationof the fused polypeptide can be encoded. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, oris an HA tag. The polynucleotide may also contain non-coding 5′ and 3′sequences, such as transcribed, non-translated sequences, splicing andpolyadenylation signals, ribosome binding sites and sequences thatstabilize mRNA.

Further preferred embodiments are polynucleotides encoding Novel 7TMreceptor (H2CAA71) variants comprising the amino acid sequence of Novel7TM receptor (H2CAA71) polypeptide of Table 2 (SEQ ID NO:2) in whichseveral, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted,deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1 or afragment thereof, may be used as hybridization probes for cDNA andgenomic DNA, to isolate full-length cDNAs and genomic clones encodingNovel 7TM receptor (H2CAA71) and to isolate cDNA and genomic clones ofother genes that have a high sequence similarity to the Novel 7TMreceptor (H2CAA71) gene. Such hybridization techniques are known tothose of skill in the art. Typically these nucleotide sequences are 80%identical, preferably 90% identical, more preferably 95% identical tothat of the referent. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding Novel 7TMreceptor (H2CAA71) polypeptide comprises the steps of screening anappropriate library under stingent hybridization condition with alabeled probe having the SEQ ID NO: 1 or a fragment thereof; andisolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Thus in another aspect, Novel 7TM receptor(H2CAA71) polynucleotides of the present invention further include anucleotide sequence comprising a nucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence having SEQ ID NO: 1or a fragment thereof. Also included with Novel 7TM receptor (H2CAA71)polypeptides are polypeptides comprising amino acid sequences encoded bya nucleotide sequence obtained by the above hybridization condition.Such hybridization techniques are well known to those of skill in theart. Stringent hybridization conditions are as defined above oralternatively conditions under overnight incubation at 42° C. in asolution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 microgram/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the Novel 7TM receptor (H2CAA71) polypeptide is to be expressed foruse in screening assays, generally, it is preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If Novel 7TM receptor(H2CAA71) polypeptide is secreted into the medium, the medium can berecovered in order to recover and purify the polypeptide; if producedintracellularly, the cells must first be lysed before the polypeptide isrecovered.

Novel 7TM receptor (H2CAA71) polypeptides can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention also relates to the use of Novel 7TM receptor (H2CAA71)polynucleotides for use as diagnostic reagents. Detection of a mutatedform of Novel 7TM receptor (H2CAA71) gene associated with a dysfunctionwill provide a diagnostic tool that can add to or define a diagnosis ofa disease or susceptibility to a disease which results fromunder-expression, over-expression or altered expression of Novel 7TMreceptor (H2CAA71). Individuals carrying mutations in the Novel 7TMreceptor (H2CAA71) gene may be detected at the DNA level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled Novel 7TM receptor (H2CAA71)nucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers et al., Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton etal., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment,an array of oligonucleotides probes comprising Novel 7TM receptor(H2CAA71) nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of e.g., genetic mutations. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability. (See for example:M. Chee et al., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome through detectionof mutation in the Novel 7TM receptor (H2CAA71) gene by the methodsdescribed.

In addition, infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, can be diagnosed by methodscomprising determining from a sample derived from a subject anabnormally decreased or increased level of Novel 7TM receptor (H2CAA71)polypeptide or Novel 7TM receptor (H2CAA71) mRNA. Decreased or increasedexpression can be measured at the RNA level using any of the methodswell known in the art for the quantitation of polynucleotides, such as,for example, PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as an Novel 7TM receptor (H2CAA71), in asample derived from a host are well-known to those of skill in the art.Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the Novel 7TM receptor (H2CAA71)polypeptides. The term “immunospecific” means that the antibodies havesubstantiall greater affinity for the polypeptides of the invention thantheir affinity for other related polypeptides in the prior art.

Antibodies generated against the Novel 7TM receptor (H2CAA71)polypeptides can be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anonhuman, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against Novel 7TM receptor (H2CAA71) polypeptides may also beemployed to treat infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with Novel 7TM receptor (H2CAA71) polypeptide, or a fragmentthereof, adequate to produce antibody and/or T cell immune response toprotect said animal from infections such as bacterial, fungal, protozoanand viral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers, anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.Yet another aspect of the invention relates to a method of inducingimmunological response in a mammal which comprises, delivering Novel 7TMreceptor (H2CAA71) polypeptide via a vector directing expression ofNovel 7TM receptor (H2CAA71) polynucleotide in vivo in order to inducesuch an immunological response to produce antibody to protect saidanimal from diseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a Novel 7TM receptor(H2CAA71) polypeptide wherein the composition comprises a Novel 7TMreceptor (H2CAA71) polypeptide or Novel 7TM receptor (H2CAA71) gene. Thevaccine formulation may further comprise a suitable carrier. Since Novel7TM receptor (H2CAA71) polypeptide may be broken down in the stomach, itis preferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradermal etc. injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in-water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The Novel 7TM receptor (H2CAA71) polypeptide of the present inventionmay be employed in a screening process for compounds which bind thereceptor and which activate (agonists) or inhibit activation of(antagonists) the receptor polypeptide of the present invention. Thus,polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixture. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See Coligan et al., Current Protocolsin Immunology 1(2):Chapter 5 (1991).

Novel 7TM receptor (H2CAA71) polypeptides are responsible for manybiological functions, including many pathologies. Accordingly, it isdesirous to find compounds and drugs which stimulate Novel 7TM receptor(H2CAA71) on the one hand and which can inhibit the function of Novel7TM receptor (H2CAA71) on the other hand. In general, agonists areemployed for therapeutic and prophylactic purposes for such conditionsas infections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome. Antagonists may be employed for avariety of therapeutic and prophylactic purposes for such conditions asinfections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome.

In general, such screening procedures involve producing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. Cells expressing the receptor (or cell membranecontaining the expressed receptor) are then contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response.

One screening technique includes the use of cells which express receptorof this invention (for example, transfected CHO cells) in a system whichmeasures extracellular pH or intracellular calcium changes caused byreceptor activation. In this technique, compounds may be contacted withcells expressing the receptor polypeptide of the present invention. Asecond messenger response, e.g., signal transduction, pH changes, orchanges in calcium level, is then measured to determine whether thepotential compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by determininginhibition or stimulation of receptor-mediated cAMP and/or adenylatecyclase accumulation. Such a method involves transfecting a eukaryoticcell with the receptor of this invention to express the receptor on thecell surface. The cell is then exposed to potential antagonists in thepresence of the receptor of this invention. The amount of cAMPaccumulation is then measured. If the potential antagonist binds thereceptor, and thus inhibits receptor binding, the levels ofreceptor-mediated cAMP, or adenylate cyclase, activity will be reducedor increased.

Another methods for detecting agonists or antagonists for the receptorof the present invention is the yeast based technology as described inU.S. Pat. No. 5,482,835 which is incorporated herein by reference.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces.Inhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed.

The Novel 7TM receptor (H2CAA71) cDNA, protein and antibodies to theprotein may also be used to configure assays for detecting the effect ofadded compounds on the production of Novel 7TM receptor (H2CAA71) mRNAand protein in cells. For example, an ELISA may be constructed formeasuring secreted or cell associated levels of Novel 7TM receptor(H2CAA71) protein using monoclonal and polyclonal antibodies by standardmethods known in the art, and this can be used to discover agents whichmay inhibit or enhance the production of Novel 7TM receptor (H2CAA71)(also called antagonist or agonist, respectively) from suitablymanipulated cells or tissues. Standard methods for conducting screeningassays are well understood in the art.

Examples of potential Novel 7TM receptor (H2CAA71) antagonists includeantibodies or, in some cases, oligonucleotides or proteins which areclosely related to the ligand of the Novel 7TM receptor (H2CAA71), e.g.,a fragment of the ligand, or small molecules which bind to the receptorbut do not elicit a response, so that the activity of the receptor isprevented.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as,infections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies; benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, related to both an excess of andinsufficient amounts of Novel 7TM receptor (H2CAA71) activity.

If the activity of Novel 7TM receptor (H2CAA71) is in excess, severalapproaches are available. One approach comprises administering to asubject an inhibitor compound (antagonist) as hereinabove describedalong with a pharmaceutically acceptable carrier in an amount effectiveto inhibit activation by clocking binding of ligands to the Novel 7TMreceptor (H2CAA71), or by inhibiting a second signal, and therebyalleviating the abnormal condition. In another approach, soluble formsof Novel 7TM receptor (H2CAA71) polypeptides still capable of bindingthe ligand in competition with endogenous Novel 7TM receptor (H2CAA71)may be administered. Typical embodiments of such competitors comprisefragments of the Novel 7TM receptor (H2CAA71) polypeptide.

In still another approach, expression of the gene encoding endogenousNovel 7TM receptor (H2CAA71) can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988) alternatively, oligonucleotides whichform triple helices with the gene can be supplied. See, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360. These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of Novel7TM receptor (H2CAA71) and its activity, several approaches are alsoavailable. One approach comprises administering to a subject atherapeutically effective amount of a compound which activates Novel 7TMreceptor (H2CAA71), i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of Novel 7TM receptor (H2CAA71) by therelevant cells in the subject. For example, a polynucleotide of theinvention may be engineered for expression in a replication defectiveretroviral vector, as discussed above. The retroviral expressionconstruct may then be isolated and introduced into a packaging celltransduced with a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention such that the packaging cell nowproduces infectious viral particles containing the gene of interest.These producer cells may be administered to a subject for engineeringcells in vivo and expression of the polypeptide in vivo. For overview ofgene therapy, see Chapter 20, Gene Therapy and other MolecularGenetic-based Therapeutic Approaches, (and references cited therein) inHuman Molecular Genetics, T Strachan and A P Read, BIOS ScientificPublishers Ltd (1996). Another approach is to administer a therapeuticamount of Novel 7TM receptor (H2CAA71) polypeptides in combination witha suitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of Novel 7TM receptor (H2CAA71)polypeptides, and agonists and antagonist peptides or small molecules,may be formulated in combination with a suitable pharmaceutical carrier.Such formulations comprise a therapeutically effective amount of thepolypeptide or compound, and a pharmaceutically acceptable carrier orexcipient. Such carriers include but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. Formulation should suit the mode of administration, and is wellwithin the skill of the art. The invention further relates topharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative mean for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples illustrate, but do not limitthe invention.

Example 1 Gene Cloning

The H2CAA71 EST (#898510) was identified from the Human Genome Sciences(HGS) database as a potential 7TM receptor and has the followingsequence:

  1 GGCACGAGAA CGCCATCATT AGGATTCACT GTAACGTTAG TGCTATTAAA (SEQ ID NO:3) 51 CTCACTAGCA TTTTTATTAA TGGCCGTTAT CTACACTAAG CTATACTGCA 101ACTTGGAAAA AGAGGACCTC TCAGAAAACT CACAATCTAG CATGATTAAG 151 CATGTCGCTTGGCTAATCTT CACCAATTGC ATCTTTTTCT GCCCTGTGGC 201 GTTTTTTTCA TTTGCACCATTGATCACTGC AATCTCTATC AGCCCCGAAA 251 TAATGAAGTC TGTTACTCTG ATATTTTTTCCATTGCCTGC TTGCCTGAAT 301 CCAGTCCTGT ATGTTTTCTT CAACCCAAAG TTTAAAGAGGACTGGGAAGT 351 TACTGAGGCG ACGTGTTTAC CAGGAAAAGT GGGTCCAGTT TCAGTTNCCN401 CATAGNCCAG GTGGTTTCTG GAACAGGGTT TNTATAGGGT TTGGGATGTA 451CTCACATTNG AAGGCAACCT GAC

This clone was ordered and sequenced completely. Analysis of thesequence revealed that the clone is a truncated clone. Therefore,oligonucleotides (5′) were designed at the 5′ end of the clone. Thisoligo was: AGTTAGGATGCVCAGTATAGATTCCC (SEQ ID NO:4). This oligo was usedto PCR a 1.3 kb 5′ fragment using the Marathon technique (Cloneteck).The 5′ PCR fragment was subcloned into pCR2.1 vector and were sequenced.This fragment was found to overlap with the original H2CAA71 truncatedclone. The full length clone is of 4.2 kb in length and it encodes aprotein of 644 amino acids.

Example 2 Mammalian Cell Expression

The receptors of the present invention are expressed in either humanembryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. Tomaximize receptor expression, typically all 5′ and 3′ untranslatedregions (UTRs) are removed from the receptor cDNA prior to insertioninto a pCDN or pCDNA3 vector. The cells are transfected with individualreceptor cDNAs by lipofectin and selected in the presence of 400 mg/mlG418. After 3 weeks of selection, individual clones are picked andexpanded for further analysis. HEK293 or CHO cells transfected with thevector alone serve as negative controls. To isolate cell lines stablyexpressing the individual receptors, about 24 clones are typicallyselected and analyzed by Northern blot analysis. Receptor mRNAs aregenerally detectable in about 50% of the G418-resistant clones analyzed.

Example 3 Ligand Bank for Binding and Functional Assays

A bank of over 200 putative receptor ligands has been assembled forscreening. The bank comprises: transmitters, hormones and chemokinesknown to act via a human seven transmembrane (7TM) receptor; naturallyoccurring compounds which may be putative agonists for a human 7TMreceptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e. calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., see below) as well as binding assays.

Example 4 Ligand Binding Assays

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for a receptor is radiolabeled to high specific activity (50-2000Ci/mmol) for binding studies. A determination is then made that theprocess of radiolabeling does not diminish the activity of the ligandtowards its receptor. Assay conditions for buffers, ions, pH and othermodulators such as nucleotides are optimized to establish a workablesignal to noise ratio for both membrane and whole cell receptor sources.For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

Example 5 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding thereceptor cDNAs of the invention are synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response to agonist exposure. Recordings are made in Ca2+ freeBarth's medium at room temperature. The Xenopus system can be used toscreen known ligands and tissue/cell extracts for activating ligands.

Example 6 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargely as a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of a receptorwhich is coupled to an energy utilizing intracellular signaling pathwaysuch as the G-protein coupled receptor of the present invention.

Example 7 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligands banks asidentified to date. Accordingly, the 7TM receptor of the invention isalso functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify natural ligands. Extracts that produce positivefunctional responses can be sequentially subfractionated until anactivating ligand is isolated identified.

Example 8 Calcium and cAMP Functional Assays

7TM receptors which are expressed in HEK 293 cells have been shown to becoupled functionally to activation of PLC and calcium mobilizationand/or cAMP stimulation or inhibition. Basal calcium levels in the HEK293 cells in receptor-transfected or vector control cells were observedto be in the normal, 100 nM to 200 nM, range, HEK 293 cells expressingrecombinant receptors are loaded with fura 2 and in a single >150selected ligands or tissue/cell extracts are evaluated for agonistinduced calcium mobilization. Similarly, HEK 293 cells expressingrecombinant receptors are evaluated for the stimulation or inhibition ofcAMP production using standard cAMP quantitation assays. Agonistspresenting a calcium transient or cAMP fluctuation are tested in vectorcontrol cells to determine if the response is unique to the transfectedcells expressing receptor.

4 1 4203 DNA HOMO SAPIENS 1 gcttccatcc taatacaact cactataggg ctcgagcggccgcccgggca ggtgcttgac 60 ggaggtgcct gtgcaccccc tcagcaatct gcccaccctacaggcgctga ccctggctct 120 caacaagatc tcaagcatcc ctgactttgc atttaccaacctttcaagcc tggtagttct 180 gcatcttcat aacaataaaa ttagaagcct gagtcaacactgttttgatg gactagataa 240 cctggagacc ttagacttga attataataa cttgggggaatttcctcagg ctattaaagc 300 ccttcctagc cttaaagagc taggatttca tagtaattctatttctgtta tccctatgga 360 gcatttgatg gtaatccact cttaagaact atacatttgtatgataatcc tctgtctttt 420 gtggggaact cagcatttca caatttatct gatcttcattccctagtcat tcgtggtgca 480 agcatggtgc agcagttccc caatcttaca ggaactgtccacctggaaag tctgactttg 540 acaggtacaa agataagcag catacctaat aatttgtgtcaagaacaaaa gatgcttagg 600 actttggact tgtcttacaa taatataaga gaccttccaagttttaatgg ttgccatgct 660 ctggaagaaa tttctttaca gcgtaatcaa atctaccaaataaaggaagg cacctttcaa 720 ggcctgatat ctctaaggat tctagatctg agtagaaacctgatacatga aattcacagt 780 agagcttttg ccacacttgg gccaataact aacctagatgtaagtttcaa tgaattaact 840 tcctttccta cggaaggcct gaatgggcta aatcaactgaaactggtggg caacttcaag 900 ctgaaagaag ccttagcagc aaaagacttt gttaacctcaggtctttatc agtaccatat 960 gcttatcagt gctgtgcatt ttggggttgt gactcttatgcaaatttaaa cacagaagat 1020 aacagcctcc aggaccacag tgtggcacag gagaaaggtactgctgatgc agcaaatgtc 1080 acaagcactc ttgaaaatga agaacatagt caaataattatccattgtac accttcaaca 1140 ggtgctttta agccctgtga atatttactg ggaagctggatgattcgtct tactgtgtgg 1200 ttcattttct tggttgcatt atttttcaac ctgcttgttattttaacaac atttgcatct 1260 tgtacatcac tgccttcgtc caaattgttt ataggcttgatttctgtgtc taacttattc 1320 atgggaatct atactggcat cctaactttt cttgatgctgtgtcctgggg cagattcgct 1380 gaatttggca tttggtggga aactggcagt ggctgcaaagtaactgggtt tcttgcagtt 1440 ttctcctcag aaagtgccat atttttatta atgctagcaactgtcgaaag aagcttatct 1500 gcaaaagata taatgaaaaa tgggaagagc aatcatctcaaacagttccg ggttgctgcc 1560 cttttggctt tcctaggtgc tacagtaaca ggctgttttccccttttcca tagaggggaa 1620 tattctgcat cacccctttg tttgccattt cctacaggtgaaacgccatc attaggattc 1680 actgtaacgt tagtgctatt aaactcacta gcatttttattaatggccgt tatctacact 1740 aagctatact gcaacttgga aaaagaggac ctctcagaaaactcacaatc tagcatgatt 1800 aagcatgtcg cttggctaat cttcaccaat tgcatctttttctgccctgt ggcgtttttt 1860 tcatttgcac cattgatcac tgcaatctct atcagccccgaaataatgaa gtctgttact 1920 ctgatatttt ttccattgcc tgcttgcctg aatccagtcctgtatgtttt cttcaaccca 1980 aagtttaaag aagactggaa gttactgaag cgacgtgttaccaagaaaag tggatcagtt 2040 tcagtttcca tcagtagcca aggtggttgt ctggaacaggatttctacta cgactgtggc 2100 atgtactcac atttgcaggg caacctgact gtttgcgactgctgcgaatc gtttctttta 2160 acaaagccag tatcatgcaa acacttgata aaatcacacagctgtcctgc attggcagtg 2220 gcttcttgcc aaagacctga gggctactgg tccgactgtggcacacagtc ggcccactct 2280 gattatgcag atgaagaaga ttcctttgtc tcagacagttctgaccaggt gcaggcctgt 2340 ggacgagcct gcttctacca gagtagagga ttccctttggtgcgctatgc ttacaatcta 2400 ccaagagtta aagactgaac tactgtgtgt gtaaccgtttcccccgtcaa ccaaaatcag 2460 tgtttataga gtgaacccta ttctcatctt tcatctgggaagcacttctg taatcactgc 2520 ctggtgtcac ttagaagaag gagaggtggc agtttatttctcaaaccagt cattttcaaa 2580 gaacaggtgc ctaaattata aattggtgaa aaatgcaatgtccaagcaat gtatgatctg 2640 tttgaaacaa atatatgact tgaaaaggat cttaggtgtagtagagcaat ataatgttag 2700 ttttttctga tccataagaa gcaaatttat acctatttgtgtattaagca caagataaag 2760 aacagctgtt aatatttttt aaaaatctat tttaaaatgtgattttctat aactgaagaa 2820 aatatcttgc taattttacc taatgtttca tccttaatctcaggacaact tactgcaggg 2880 ccaaaaaagg gactgtccca gctagaactg tgagagtatacataggcatt actttattat 2940 gttttcactt gccatccttg acataagaga actataaattttgtttaagc aatttataaa 3000 tctaaaacct gaagatgttt ttaaaacaat attaacagctgttaggttaa aaaaatagct 3060 ggacatttgt tttcagtcat tatacattgc tttggtccaatcagtaattt tttcttaagt 3120 gttttgtgat tacactacta gaaaaaaagt aaaaggctaattgctgtgtg ggtttagtcg 3180 atttggctaa actactaact aatgtggggg tttaatagtatctgagggat ttggtggctt 3240 catgtaatgt tctcattaat gaatacttcc taatatcgttggctctacta atattttcca 3300 atttgctggg atgtcaccta gcaatagctt ggattatatagaaagtaaac tgtggtcaat 3360 acttgcattt aattagacga aacggggagt aattatgacacgaagtactt atgtttattt 3420 cttagtgagc tggattatct tgaacctgtg ctattaaatggaaatttcca tacatcttcc 3480 ccatactatt ttttataaaa gagcctattc aatagctcagaggttgaact ctggttaaac 3540 aagataatat gttattaata aaaatagaag aagaaagaataaagcttagt cctgtgtctt 3600 taaaaattaa aaattttact tgattcccat ctatgggctttagacctatt actgggtgga 3660 gtcttaaagt tataattgtt caatatgttt tttgaacagtgtgctaaatc aatagcaaac 3720 ccactgccat attagttatt ctgaatatac taaaaaaatccagctagatt gcagtttaat 3780 aattaaactg tacatactgt gcatataatg aatttttatcttatgtaaat tatttttaga 3840 acacaagttg ggaaatgtgg cttctgttca tttcgtttaattaaagctac ctcctaaact 3900 atagtggctg ccagtagcag actgttaaat tgtggtttatatactttttg cattgtaaat 3960 agtctttgtt gtacattgtc agtgtaataa aaacagaatctttgtatatc aaaatcatgt 4020 agtttgtata aaatgtggga aggatttatt tacagtgtgttgtaattttg taaggccaac 4080 tatttacaag ttttaaaaat tgctatcatg tatatttacacatctgataa atattaaatc 4140 ataacttggt aagaaactcc taattaaaag gttttttccaaaaaaaaaaa aaaaaaaaaa 4200 aaa 4203 2 644 PRT HOMO SAPIENS 2 Met Val GlnGln Phe Pro Asn Leu Thr Gly Thr Val His Leu Glu Ser 1 5 10 15 Leu ThrLeu Thr Gly Thr Lys Ile Ser Ser Ile Pro Asn Asn Leu Cys 20 25 30 Gln GluGln Lys Met Leu Arg Thr Leu Asp Leu Ser Tyr Asn Asn Ile 35 40 45 Arg AspLeu Pro Ser Phe Asn Gly Cys His Ala Leu Glu Glu Ile Ser 50 55 60 Leu GlnArg Asn Gln Ile Tyr Gln Ile Lys Glu Gly Thr Phe Gln Gly 65 70 75 80 LeuIle Ser Leu Arg Ile Leu Asp Leu Ser Arg Asn Leu Ile His Glu 85 90 95 IleHis Ser Arg Ala Phe Ala Thr Leu Gly Pro Ile Thr Asn Leu Asp 100 105 110Val Ser Phe Asn Glu Leu Thr Ser Phe Pro Thr Glu Gly Leu Asn Gly 115 120125 Leu Asn Gln Leu Lys Leu Val Gly Asn Phe Lys Leu Lys Glu Ala Leu 130135 140 Ala Ala Lys Asp Phe Val Asn Leu Arg Ser Leu Ser Val Pro Tyr Ala145 150 155 160 Tyr Gln Cys Cys Ala Phe Trp Gly Cys Asp Ser Tyr Ala AsnLeu Asn 165 170 175 Thr Glu Asp Asn Ser Leu Gln Asp His Ser Val Ala GlnGlu Lys Gly 180 185 190 Thr Ala Asp Ala Ala Asn Val Thr Ser Thr Leu GluAsn Glu Glu His 195 200 205 Ser Gln Ile Ile Ile His Cys Thr Pro Ser ThrGly Ala Phe Lys Pro 210 215 220 Cys Glu Tyr Leu Leu Gly Ser Trp Met IleArg Leu Thr Val Trp Phe 225 230 235 240 Ile Phe Leu Val Ala Leu Phe PheAsn Leu Leu Val Ile Leu Thr Thr 245 250 255 Phe Ala Ser Cys Thr Ser LeuPro Ser Ser Lys Leu Phe Ile Gly Leu 260 265 270 Ile Ser Val Ser Asn LeuPhe Met Gly Ile Tyr Thr Gly Ile Leu Thr 275 280 285 Phe Leu Asp Ala ValSer Trp Gly Arg Phe Ala Glu Phe Gly Ile Trp 290 295 300 Trp Glu Thr GlySer Gly Cys Lys Val Thr Gly Phe Leu Ala Val Phe 305 310 315 320 Ser SerGlu Ser Ala Ile Phe Leu Leu Met Leu Ala Thr Val Glu Arg 325 330 335 SerLeu Ser Ala Lys Asp Ile Met Lys Asn Gly Lys Ser Asn His Leu 340 345 350Lys Gln Phe Arg Val Ala Ala Leu Leu Ala Phe Leu Gly Ala Thr Val 355 360365 Thr Gly Cys Phe Pro Leu Phe His Arg Gly Glu Tyr Ser Ala Ser Pro 370375 380 Leu Cys Leu Pro Phe Pro Thr Gly Glu Thr Pro Ser Leu Gly Phe Thr385 390 395 400 Val Thr Leu Val Leu Leu Asn Ser Leu Ala Phe Leu Leu MetAla Val 405 410 415 Ile Tyr Thr Lys Leu Tyr Cys Asn Leu Glu Lys Glu AspLeu Ser Glu 420 425 430 Asn Ser Gln Ser Ser Met Ile Lys His Val Ala TrpLeu Ile Phe Thr 435 440 445 Asn Cys Ile Phe Phe Cys Pro Val Ala Phe PheSer Phe Ala Pro Leu 450 455 460 Ile Thr Ala Ile Ser Ile Ser Pro Glu IleMet Lys Ser Val Thr Leu 465 470 475 480 Ile Phe Phe Pro Leu Pro Ala CysLeu Asn Pro Val Leu Tyr Val Phe 485 490 495 Phe Asn Pro Lys Phe Lys GluAsp Trp Lys Leu Leu Lys Arg Arg Val 500 505 510 Thr Lys Lys Ser Gly SerVal Ser Val Ser Ile Ser Ser Gln Gly Gly 515 520 525 Cys Leu Glu Gln AspPhe Tyr Tyr Asp Cys Gly Met Tyr Ser His Leu 530 535 540 Gln Gly Asn LeuThr Val Cys Asp Cys Cys Glu Ser Phe Leu Leu Thr 545 550 555 560 Lys ProVal Ser Cys Lys His Leu Ile Lys Ser His Ser Cys Pro Ala 565 570 575 LeuAla Val Ala Ser Cys Gln Arg Pro Glu Gly Tyr Trp Ser Asp Cys 580 585 590Gly Thr Gln Ser Ala His Ser Asp Tyr Ala Asp Glu Glu Asp Ser Phe 595 600605 Val Ser Asp Ser Ser Asp Gln Val Gln Ala Cys Gly Arg Ala Cys Phe 610615 620 Tyr Gln Ser Arg Gly Phe Pro Leu Val Arg Tyr Ala Tyr Asn Leu Pro625 630 635 640 Arg Val Lys Asp 3 473 DNA HOMO SAPIENS UNSURE(397)(400)(406)(432)(459) 3 ggcacgagaa cgccatcatt aggattcact gtaacgttagtgctattaaa ctcactagca 60 tttttattaa tggccgttat ctacactaag ctatactgcaacttggaaaa agaggacctc 120 tcagaaaact cacaatctag catgattaag catgtcgcttggctaatctt caccaattgc 180 atctttttct gccctgtggc gtttttttca tttgcaccattgatcactgc aatctctatc 240 agccccgaaa taatgaagtc tgttactctg atattttttccattgcctgc ttgcctgaat 300 ccagtcctgt atgttttctt caacccaaag tttaaagaggactgggaagt tactgaggcg 360 acgtgtttac caggaaaagt gggtccagtt tcagttnccncatagnccag gtggtttctg 420 gaacagggtt tntatagggt ttgggatgta ctcacattngaaggcaacct gac 473 4 25 DNA HOMO SAPIENS 4 agttaggatg ccagtataga ttccc25

What is claimed is:
 1. A Novel 7TM receptor (H2CAA71) polypeptidecomprising an amino acid sequence which is at least 80% identical to theamino acid sequence of SEQ ID NO:2 over its entire length, said identityis calculated using FASTA, setting parameters such that the highestorder match is obtained.
 2. The polypeptide of claim 1 which comprisesthe amino acid sequence set forth in SEQ ID NO:2.